SUMMARY Excluding the great apes, the immune system of Indian rhesus macaques (RMs) most closely approximates that of humans. Thus, it is widely believed that the RM represents the closest practical animal model for evaluating protective immunity to HIV-1 and vaccines that might elicit such protection. For the analysis of HIV-1 neutralizing antibodies (NAbs), RMs have in the past been infected with simian-human immunodeficiency viruses (SHIVs) that bear HIV-1 Envs that were heavily adapted in cell culture or by animal passage, or they were immunized with any of numerous Env containing candidate vaccines ranging from Env peptides or DNA vectors to gp120 monomers to soluble stabilized Env trimers (SOSIPs). The findings from all such studies to date have been the same: Abs can be generated that neutralize heterologous tier 1 viruses, and in some instances autologous tier 2 viruses, but generally not heterologous tier 2 viruses that constitute virtually all primary HIV-1 strains and are responsible for clinical HIV-1 transmission. Surprisingly, there is no direct experimental evidence that RMs have the potential to develop heterologous tier 2 broadly neutralizing antibodies (bNAbs) even if vaccinated with an appropriate immunogen. Thus, a major roadblock to HIV-1 vaccine development is that there is no animal model system where the elicitation of HIV-1 bNAbs can be reliably and consistently demonstrated, let alone be studied in an iterative fashion for rational vaccine design. Project 1 seeks to overcome this roadblock by capitalizing on recent discoveries by the Shaw lab regarding novel SHIV design strategies. The scientific premise of Project 1 is that since most examples of bNAb elicitation come from natural human infection by primary strains of HIV-1, the most likely means to elicit bNAbs in RMs is by infection with SHIVs bearing primary HIV-1 Envs in their native conformations. A key hypothesis that we will test is that SHIVs bearing primary or transmitted/founder (T/F) Envs will engage rhesus germline and intermediate ancestor B cell receptors (BCRs) that are orthologous to those engaged by the same Envs in human infection and that patterns of virus-Ab coevolution leading to the development of neutralizing antibody specificity, potency and breadth will be similar in both species. Specific Aims of Project 1 are to: (i) analyze in 33 RMs the in vivo replication kinetics and NAb responses of 11 novel SHIVs whose Envs are known to bind to human bNAb lineage BCRs targeting the V3-glycan supersite (CH848, CH694, CH040), V1V2 supersite (CAP256SU, ZM233, WITO, Q23, T250-4, CH1012), CD4bs (CH505) or other (BG505), and down-select to two SHIVs for inoculation into 9 RMs each; (ii) analyze in vivo replication kinetics and env evolutionary pathways of SHIVs targeting V3-glycan or V1V2 germline and IA BCRs; and (iii) determine the kinetics, potency and breadth of polyclonal NAb responses and map epitope specificities by a combination of Env fingerprinting, Env-expression cloning, and site-directed mutagenesis. Project 1 will link closely with Cores B and C for sequencing and bioinformatics and Projects 2 and 3 for sample provision and data interpretation.